Paf1 complex subunit Rtf1 stimulates H2B ubiquitylation by interacting with the highly conserved N-terminal helix of Rad6
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA901941 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR22301100_1.fastq.gz | Fastqsanger.gz | |||
| SRR22301100_2.fastq.gz | Fastqsanger.gz | |||
| SRR22301101_1.fastq.gz | Fastqsanger.gz | |||
| SRR22301101_2.fastq.gz | Fastqsanger.gz | |||
| SRR22301102_1.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 48 |
Similar Datasets
Project description:Histone modifications coupled to transcription elongation play important roles in regulating the accuracy and efficiency of gene expression. The mono-ubiquitylation of a conserved lysine in H2B (K123 in Saccharomyces cerevisiae and K120 in humans) occurs co-transcriptionally and is required for initiating a histone modification cascade on active genes. H2BK123 ubiquitylation (H2BK123ub) requires the RNA polymerase II (RNAPII)-associated Paf1 transcription elongation complex (Paf1C). Through its Histone Modification Domain (HMD), the Rtf1 subunit of Paf1C directly interacts with the ubiquitin conjugase Rad6, leading to the stimulation of H2BK123ub in vivo and in vitro. To understand the molecular mechanisms that specifically target Rad6 to its histone substrate, we identified the site of interaction for the HMD on Rad6. Using in vitro crosslinking followed by mass spectrometry, we localized the primary contact surface for the HMD to the highly conserved N-terminal helix of Rad6. Using a combination of genetic and biochemical experiments, we identified separation-of-function mutations in RAD6 that greatly impair H2BK123 ubiquitylation but not other Rad6 functions. Finally, by employing RNA-sequencing as a sensitive approach for comparing mutant phenotypes, we show that mutating either side of the proposed Rad6-HMD interface yields strikingly similar transcriptome profiles that extensively overlap with those of a mutant that lacks the site of ubiquitylation in H2B. Our results fit a model in which a specific interface between a transcription elongation factor and a ubiquitin conjugase guides substrate selection toward a highly conserved chromatin target during active gene expression.
2023-05-01 | GSE218023 | GEO
Project description:Transcription by RNA polymerase II is regulated by epigenetic modifications to the chromatin template. The mono-ubiquitylation of H2B is established during transcription elongation and broadly impacts chromatin architecture and gene expression. The Polymerase Associated Factor 1 complex (Paf1C) is required for H2B ubiquitylation through an unknown mechanism. Here, we find that a 66-amino acid histone modification domain (HMD) within the Rtf1 subunit of Paf1C promotes H2B ubiquitylation in cells lacking all Paf1C members and present the crystal structure of this domain. Using site-specific in vivo crosslinking, we show that Rtf1 directly interacts with the ubiquitin conjugase Rad6 through a conserved surface on the HMD. Through ChIP-exo analysis, we observe that enrichment of Paf1C correlates with H2B ubiquitylation, and that the HMD, Rad6 and Bre1 localize to H2B. Finally, we demonstrate that the HMD directly stimulates H2B ubiquitylation in a reconstituted system, arguing that Paf1C functions as a cofactor for Rad6-Bre1 mediated catalysis.
2016-11-07 | GSE83348 | GEO
Project description:Histone modifications coupled to transcription elongation play important roles in regulating the accuracy and efficiency of gene expression. The monoubiquitylation of a conserved lysine in H2B (K123 in Saccharomyces cerevisiae; K120 in humans) occurs cotranscriptionally and is required for initiating a histone modification cascade on active genes. H2BK123 ubiquitylation (H2BK123ub) requires the RNA polymerase II (RNAPII)-associated Paf1 transcription elongation complex (Paf1C). Through its histone modification domain (HMD), the Rtf1 subunit of Paf1C directly interacts with the ubiquitin conjugase Rad6, leading to the stimulation of H2BK123ub in vivo and in vitro. To understand the molecular mechanisms that target Rad6 to its histone substrate, we identified the site of interaction for the HMD on Rad6. Using in vitro cross-linking followed by mass spectrometry, we localized the primary contact surface for the HMD to the highly conserved N-terminal helix of Rad6. Using a combination of genetic, biochemical, and in vivo protein cross-linking experiments, we characterized separation-of-function mutations in S. cerevisiae RAD6 that greatly impair the Rad6-HMD interaction and H2BK123 ubiquitylation but not other Rad6 functions. By employing RNA-sequencing as a sensitive approach for comparing mutant phenotypes, we show that mutating either side of the proposed Rad6-HMD interface yields strikingly similar transcriptome profiles that extensively overlap with those of a mutant that lacks the site of ubiquitylation in H2B. Our results fit a model in which a specific interface between a transcription elongation factor and a ubiquitin conjugase guides substrate selection toward a highly conserved chromatin target during active gene expression.
| S-EPMC10235976 | biostudies-literature
Project description:The five-subunit yeast Paf1 complex (Paf1C) regulates all stages of transcription and is critical for the monoubiquitylation of histone H2B (H2Bub), a modification that broadly influences chromatin structure and eukaryotic transcription. Here, we show that the histone modification domain (HMD) of Paf1C subunit Rtf1 directly interacts with the ubiquitin conjugase Rad6 and stimulates H2Bub independently of transcription. We present the crystal structure of the Rtf1 HMD and use site-specific, in vivo crosslinking to identify a conserved Rad6 interaction surface. Utilizing ChIP-exo analysis, we define the localization patterns of the H2Bub machinery at high resolution and demonstrate the importance of Paf1C in targeting the Rtf1 HMD, and thereby H2Bub, to its appropriate genomic locations. Finally, we observe HMD-dependent stimulation of H2Bub in a transcription-free, reconstituted in vitro system. Taken together, our results argue for an active role for Paf1C in promoting H2Bub and ensuring its proper localization in vivo.
| S-EPMC5131541 | biostudies-literature
Project description:Paf1 and Ski8 were selected as representative subunits of the Paf1 complex (PAF1C), and RNA-seq analysis was performed in triplicate to compare the genes affected by Paf1, Ski8, and Rtf1 knockdown in HeLa cells.
2015-10-01 | GSE73632 | GEO
Project description:RNA polymerase II (RNAPII) transcription elongation directs an intricate pattern of histone modifications. Formation of this pattern involves a highly conserved regulatory axis, key components of which are the elongation factor Rtf1, monoubiquitylation of histone H2B (H2Bub1), and methylation of histone H3 on lysine 4 (H3K4me). Direct contact between Rtf1 and the ubiquitylation complex Rad6-Bre1 stimulates H2Bub1, which in turn promotes H3K4me by stimulating the activity of the Set1C/COMPASS methyltransferase complex. Previous studies have defined the molecular basis for these regulatory relationships, but it remains unclear how they regulate chromatin and transcription in vivo. We found that mutations affecting multiple components of the Rtf1-H2Bub1-H3K4me axis in the model eukaryote Schizosaccharomyces pombe caused resistance to the ribonucleotide reductase inhibitor hydroxyurea (HU). Resistance correlated with a reduced effect of HU on dNTP pools, bypass of the S-phase checkpoint in the presence of HU, and blunting of the transcriptional response to HU treatment. Mutations in the C-terminal repeat domain (CTD) of the RNAPII large subunit Rpb1 led to a similar spectrum of phenotypes. Moreover, all the HU resistant mutants we identified also exhibited resistance to several azole class antifungal agents. Our results suggest a novel, shared gene regulatory function of the Rtf1-H2Bub1-H3K4me axis and the Rpb1 CTD in controlling fungal drug tolerance.
2023-11-27 | GSE248361 | GEO
Project description:RNA polymerase II (RNAPII) transcription elongation directs an intricate pattern of histone modifications. Formation of this pattern involves a highly conserved regulatory axis, key components of which are the elongation factor Rtf1, monoubiquitylation of histone H2B (H2Bub1), and methylation of histone H3 on lysine 4 (H3K4me). Direct contact between Rtf1 and the ubiquitylation complex Rad6-Bre1 stimulates H2Bub1, which in turn promotes H3K4me by stimulating the activity of the Set1C/COMPASS methyltransferase complex. Previous studies have defined the molecular basis for these regulatory relationships, but it remains unclear how they regulate chromatin and transcription in vivo. We found that mutations affecting multiple components of the Rtf1-H2Bub1-H3K4me axis in the model eukaryote Schizosaccharomyces pombe caused resistance to the ribonucleotide reductase inhibitor hydroxyurea (HU). Resistance correlated with a reduced effect of HU on dNTP pools, bypass of the S-phase checkpoint in the presence of HU, and blunting of the transcriptional response to HU treatment. Mutations in the C-terminal repeat domain (CTD) of the RNAPII large subunit Rpb1 led to a similar spectrum of phenotypes. Moreover, all the HU resistant mutants we identified also exhibited resistance to several azole class antifungal agents. Our results suggest a novel, shared gene regulatory function of the Rtf1-H2Bub1-H3K4me axis and the Rpb1 CTD in controlling fungal drug tolerance.
2023-11-27 | GSE248360 | GEO
Project description:Monodelphis domestica RTF1, RTF1 homolog, Paf1/RNA polymerase II complex component [Source:NCBI gene;Acc:100032121], is expressed in 2 baseline experiment(s);
| ENSMODG00000000135 | ExpressionAtlas
Project description:Mus musculus Rtf1, RTF1, Paf1/RNA polymerase II complex component [Source:MGI Symbol;Acc:MGI:1309480], is differentially expressed in 65 experiment(s);
| ENSMUSG00000027304 | ExpressionAtlas
Project description:Mus musculus Rtf1, RTF1, Paf1/RNA polymerase II complex component [Source:MGI Symbol;Acc:MGI:1309480], is expressed in 44 baseline experiment(s);
| ENSMUSG00000027304 | ExpressionAtlas